Structural and functional insights into the bona fide catalytic state of Streptococcus pyogenes Cas9 HNH nuclease domain
- Shanghai University of Engineering Science, China
- University of North Texas Health Science Center, United States
- University of Oklahoma, United States
Abstract
The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (SpyCas9), along with a programmable single-guide RNA (sgRNA), has been exploited as a significant genome-editing tool. Despite the recent advances in determining the SpyCas9 structures and DNA cleavage mechanism, the cleavage-competent conformation of the catalytic HNH nuclease domain of SpyCas9 remains largely elusive and debatable. By integrating computational and experimental approaches, we unveiled and validated the activated Cas9-sgRNA-DNA ternary complex in which the HNH domain is neatly poised for cleaving the target DNA strand. In this catalysis model, the HNH employs the catalytic triad of D839-H840-N863 for cleavage catalysis, rather than previously implicated D839-H840-D861, D839-H840-N854, D837-D839-H840, or D839-H840-D861-N863. Our study contributes critical information to defining the catalytic conformation of the HNH domain and advances the knowledge about the conformational activation underlying Cas9-mediated DNA cleavage.
Data availability
The data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1 and 2.
Article and author information
Author details
Hamed S Hayatshahi
Funding
Shanghai Municipal Education Commission (Program for Professor of Special Appointment at Shanghai Institutions of Higher Learning)
- Zhicheng Zuo
National Science Foundation (MCB-1716423)
- Rakhi Rajan
National Institute of General Medical Sciences (P20GM103640)
- Rakhi Rajan
University of North Texas Health Science Center (Start-up Fund and Faculty Pilot Grant)
- Yu-Chieh Wang
University of North Texas Health Science Center (Start-up Fund and Basic Research Seed Grant)
- Jin Liu
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Zuo et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Structural and functional insights into the bona fide catalytic state of Streptococcus pyogenes Cas9 HNH nuclease domain
eLife 8:e46500.
https://doi.org/10.7554/eLife.46500
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